We are alleviating the toxicity of the anticancer drug CPT-11 by modulating components of the GI microbiome. CPT-11 is essential in treating colorectal and pancreatic cancer, but dose-limiting toxicity severely reduces its efficacy. This toxicity is caused by a bacterial enzyme in enteric microbial symbiotes. The enzyme, beta glucuronidase, removes the inactivating glucuronic acid sugar from CPT-11's key metabolite, which reactivates the drug in the GI and produces epithelial cell death and acute diarrhea. We hypothesized that the selective, non-lethal inhibition of microbial beta glucuronidases would alleviate this side effect. This hypothesis tested true in proof-of-concept molecular-to-animal studies conducted in the previous project period. We will now advance the project in three crucial ways. First, we will characterize the range of active ?-glucuronidases present in the GI microbiome using structural biology and biochemistry. Second, we will create differentially optimized bacterial beta glucuronidase inhibitors via structural and chemical biology. Third, using deep-sequencing and metagenomics, we will unravel how this approach impacts the composition and activity of the GI microbiome. In summary, we seek to advance a novel paradigm - inhibiting specific microbial enzymes for therapeutic gain without harming the bacterial symbiotes essential for human health.